Fluorescence spectroscopy of macromolecules is used in many areas of biology. Application of the technique can produce valuable information due to the inherently high sensitivity of fluorescence emission and the abundance of potential labeling targets in cells. Types of questions that can be addressed include the identification of antigens on cell surfaces, the study of subcellular compartmentalization, and the dynamics of macromolecular motion. While many of these applications require only that a molecule be labeled with a fluorescent probe at a sufficiently high level to visualize it, other applications, for example, the measurement of rotational diffusion of membrane proteins, depend critically upon the uniformity of protein labeling and the extent to which the dynamic properties of a fluorescent group accurately reflect the dynamics of the target molecules.
Typically, proteins such as antibodies are labeled on the nucleophilic sites on the surface of the protein, i.e. the amino chain termini and the epsilon-amino groups of lysines, using fluorophores containing reactive groups such as isothiocyanates, triazinates, and sulfonyl chlorides. Additionally, the sulfhydryls of cysteines as well as the carboxyls of aspartic and glutamic acids are potential targets for labeling with fluorophores. Several drawbacks are inherent in all commonly used covalent labeling procedures. First, proteins contain a multitude of comparably reactive groups; thus, labeling of proteins usually results in a population of molecules with a random distribution of label location and substitution level. Unfortunately, many of the labeled sites on a protein exhibit sufficient chemical homogeneity that resolution into distinct species is not readily feasible. Thus, even if the substitution level of fluorophore to protein is 1:1, multiple fluorescent environments may coexist. Hence, if the degree of label incorporation is not uniform, proteins labeled in this manner would not be desirable for use in a quantitative assay since the range of fluorescence signals for a given fixed antigen could vary greatly.
A second drawback is the autonomous rotational motion that a fluorophore may retain relative to the labeled protein molecule. This is a consequence of the fact that the chemical linkage between commonly used fluorophores and proteins is a single covalent bond. For many classes of measurements, this is not a drawback; however if, for example, one uses a fluorescence signal to determine the rotational motion of a protein, the time-dependent polarization may be dominated by the motion of the probe. Thus, the value obtained is probably not a true indicator of the rotational correlation time of the protein.
A third drawback is that the commonly used method of labeling the surface of the protein may result in a modified tertiary structure which can alter the biological activity of the protein.
Generally, in the dye industry, the dye molecule is hydrophobic and associates with the fibrous protein such as hair, wool, silk, etc., in a non covalent complex. A problem associated with this method of coloring materials with dye is that over time the dye fades, as the dye molecules dissociate from the fibrous protein. Other methods of dyeing fibrous proteins include the attachment of the dye molecule to the fibrous protein by a single covalent bond created by alkylation, or using one cysteine group. This process is irreversible.
The drug delivery systems presently used involve attachment of a drug to the surface of a protein such as an antibody creating an antibody conjugate. This method, however, can cause an immune system response since the conjugated antibody is recognized by the body as a foreign molecule.
Molecules with bifunctional groups have been attached to proteins, e.g. where one functional group reacts with a sulfhydryl of the protein while the other reacts with an amino group of the protein. This bifunctional method of modifying the protein, however, is not reversible and can change the tertiary structure of the protein. If only one of the --SH groups of a reduced disulfide bond is reacted, the structural stabilizing influence of the original --S--S bond is lost and the tertiary structure is likely to change.